Proteomic and Functional Analyses of Astrocyte Exosomes Astrocytes are the most abundant cell type in the central nervous system (CNS). Over the past decade increasing attention has been paid to the critical homeostatic and diverse roles astrocytes play in the CNS health and disease. The emerging influence of astrocytes in CNS physiology and pathology has been highlighted by how factors secreted by astrocytes impact CNS development, synaptic transmission, neurodegeneration, or infection. Hence, understanding how astrocytes respond to and are regulated by extracellular cues is expected to identify fundamental mechanisms that govern how the CNS adapts and responds to injury or inflammation. Exosomes are a class of very small extracellular vesicles that are known to be secreted by many cell types. Recent studies have now identified critical functions for exosomes in mediating cell signaling and inflammatory responses. However, little is known about the function of exosomes released from astrocytes in response to inflammation and no previous study has detailed what proteins are contained in astrocytic exosomes. This gap in our knowledge will be addressed in this application as follows: Specific Aim 1 will generate a proteomic map of the proteins contained within exosomes isolated from astrocytes, under basal and inflammatory conditions, using a cutting edge proteomic fractionation platform. Specific Aim 2 will determine the function of astrocyte exosomes as putative mediators of astrocytic responses to inflammatory stimuli both in vitro and in vivo. We provide preliminary data that demonstrate a qualitative and quantitative increase in the release of exosomes from cultured astrocytes in response to treatment with interleukin-1beta. Importantly, we present data to indicate that astrocyte-derived exosomes are present within systemic circulation and can be collected and quantified from blood serum. We hypothesize that exosomes represent a fundamental component of the astrocytic response to inflammatory and neuropathological conditions. Results from these studies will provide a new functional basis to understand how exosomes define astrocyte function. This work will provide the scientific community with two shared outcomes: (1) we will produce a proteomic map of astrocytic exosomes under basal and inflammatory conditions that might lead to high fidelity biomarker identification, and (2) we will determine the functional role of astrocytic exosomes in in vitro and in vivo models. Thus, these studies may stimulate widespread interest in astrocyte-derived exosomes as mediators of intercellular communication in the CNS in health and disease.